For transparent and semitransparent objects, mainly in the life sciences, a microscope with internal light transmission is normally used. To make objects visible, the four most important optical effects are: absorption, scattering, phase shifting and fluorescence.
The convolution described herein can be used for objects with the properties of absorption, scattering, a gradient of refractive index and fluorescence. However, important practical applications are with the optical effects of absorption and scattering and this description focuses on microscopes used for absorbing and scattering objects.
FIG. 1 shows a prior art system in which a microscope transmits light from a light-emitting filament 20 through a collector lens 21. The collector lens causes the light to be bent in a manner such that parallel beams, such as beams 18 and 19, are caused to intersect each other in the plane of a radiant field stop 22. Light then passes through an aperture field stop 23. A condenser lens 24 focuses the illuminating light onto an object 25 to be magnified. This object is typically a transparent object with light absorbing regions and scattering regions such as biological cells or tissues. These cells or tissues are typically placed on a pane of glass and covered with another very small and thin pane of glass. This arrangement is referred to as a microscope slide or object or specimen. The light not absorbed by the object 25 enters an objective 26 and then passes through an objective output pupil 27 and is focused onto a plane where a target 28 of a TV camera tube is located. The TV camera tube captures the images for the microscope. A normal microscope with eyepieces has a field stop at location 28 which is followed by a binocular. The present invention is not intended for this type of microscope because the images would be deteriorated by the arrangements of this invention.
There is an important fact concerning the light path shown between the condenser lens 24 and the objective 26. The light of the two original parallel beams, shown as dotted lines 18 and 19, from the filament 20 defines a double cone 29 which intersects in the object 25. There exist as many double cones as there are pixels in an optical image. Each of the more than 100,000, typically a million, double cones stem from parallel light emitted by the filament 20. The image projected to the TV tube target 28 is a convolution of the object 25. As shown in FIG. 1, the apexes 30 of the double cones form a plane. Only those details of the objects which lie in this plane are in focus. The further away the details of the object are from the focus plane, the less the details can be recognized in the image that is projected to the TV target.
State-of-the-art microscopes have a manually operated gear which moves either the object 25 or the objective 26 or a lens in the objective in the direction of the axis of the system in order to focus on successive planes of the object. However, the image contains not only the details in the focal plane but also the defocused details of other parts of the object. This leads to severe disadvantages from which all light microscopes suffer. As the object becomes thicker, the resolution achieved for details in the focal plane deteriorates. This situation is exacerbated when stained objects contain many details distributed over a large volume.
To overcome this problem in the field of biological research and other sciences, specimens are cut into very thin layers using microtomes. This is a time-consuming method, often needing refrigeration or freezing of the object before cutting, and it cannot be applied to achieve images from living cells. Therefore, computers have been used to convolute sequences of microscopic images which have been stored in computer memory by stepping from one focal point to the next. Detail with respect to the foregoing are provided in U.S. Pat. No. 4,360,885 incorporated herein by reference. Additional details on the above are described in literature such as Ehrhard, Zinser, Komitowski, Bille, Reconstructing 3-D Light-Microscope Images by Digital Image Processing, Applied Optics, Vol. 24 pp. 194-200. The convolution can be done by applying a three-dimensional Fourier transform to a cube of 64.times.64.times.64 pixels with eight bits each. This yields a new set of 64.times.64.times.64 pixels, but now in frequency domain. It may be called the Fourier data cube. By the same method the double cone is also transformed into frequency domain. It may be called the Fourier instrument function. The reciprocal of the Fourier instrument function is computed and multiplied with the Fourier data cube. The result of this product of the image data and the reciprocal of the instrument function computed in frequency domain is the improved image data still in frequency domain. It then is treated with the inverse Fourier transform. The result is the desired data of the specimen improved by convolution.
This is a very powerful mathematical method. However, the results are discouraging because the images displayed on a TV screen after computation are not as detailed as needed for practical applications and because too much noise deteriorates the images.
A phase contrast microscope, such as that shown is U.S. Pat. No. 2,660,923, looks similar to the invention which will be described later. Phase contrast microscopes are built to generate images from details of objects which are differentiated by their phase shifting details as opposed to absorption or scattering characteristics. The microscope as shown in FIG. 1 would not show the phase shifting details. The microscope of FIG. 1 has an aperture field stop 23 and an objective outlet pupil 27 which are both shaped as shown in FIG. 2. To adapt this microscope to show phase shifting properties, it is equipped with a ring-shaped aperture field stop as shown in FIG. 3 and a ring-structured objective outlet pupil as shown in FIG. 4. In both figures dark areas 22 indicate opaque regions, light areas 24 indicate 100% transparent regions and the gray-shaded area 20 in FIG. 4 indicates transparency of about 20% plus an angle shift of the light of 90 degrees. The lenses of the phase contrast microscope are designed such that the transparent ring structure from FIG. 3 is focused to the gray-shaded ring in FIG. 4, both being the same size. Thus, the direct light from the light source 20 interferes with the light diffracted by the specimen on the TV target 28. Microscopes of this type show images of different details of the object as dark and light regions. In contrast to the invention described herein, the purpose of the rings shown in FIGS. 2 and 3 is to provide different light paths for the direct and diffracted light and to shift the phase of the direct light. Annular apertures and annular structures in the light path also are disclosed for different purposes in British Pat. No. 1,595,422, in U.S. Pat. Nos. 2,660,923, 4,150,360 and 4,202,037 incorporated herein by this reference.